U.S. patent number 6,143,570 [Application Number 09/307,914] was granted by the patent office on 2000-11-07 for optical sensor for the determination of ions.
This patent grant is currently assigned to Novartis Corporation. Invention is credited to Alex Alder, Steven Barnard, Joseph Berger.
United States Patent |
6,143,570 |
Alder , et al. |
November 7, 2000 |
Optical sensor for the determination of ions
Abstract
A composition comprising (a) a transparent support (b) which is
coated on at least one side with a transparent coating which
comprises (b1) a plasticizer-free, hydrophobic polymer having a
glass transition temperature Tg of from -150 to 50.degree. C., (b2)
counterions in the form of lipophilic salts, (b3) an ionophore
which forms a complex with the ion to be determined, and (b4) a
compound of the formula I or II as fluorophore ##STR1## in which
R.sub.1 and R.sub.3, and R.sub.4 and R.sub.6 are C.sub.1 -C.sub.30
alkyl or C.sub.1 -C.sub.30 alkyl-CO--, and R.sub.2 and R.sub.5 are
H or C.sub.1 -C.sub.30 alkyl, with the proviso that the total
number of carbon atoms in the alkyl groups is at least 5, or a salt
thereof with an inorganic or organic acid. The composition is
highly suitable for the qualitative or quantitative optical
determination of ions by fluorescence detection.
Inventors: |
Alder; Alex (Arisdorf,
CH), Barnard; Steven (Wellesley, MA), Berger;
Joseph (Basel, CH) |
Assignee: |
Novartis Corporation (Summit,
NJ)
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Family
ID: |
4198183 |
Appl.
No.: |
09/307,914 |
Filed: |
May 10, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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704703 |
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Foreign Application Priority Data
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Mar 25, 1994 [CH] |
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917/94-0 |
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Current U.S.
Class: |
436/74;
422/82.08; 436/172; 436/79; 422/429 |
Current CPC
Class: |
C09B
11/22 (20130101); G01N 31/221 (20130101); C09B
15/00 (20130101); G01N 2410/10 (20130101) |
Current International
Class: |
C09B
15/00 (20060101); C09B 11/22 (20060101); C09B
11/00 (20060101); G01N 31/22 (20060101); G01N
021/64 () |
Field of
Search: |
;422/82.08,57
;436/73,74,79,172 |
References Cited
[Referenced By]
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4549951 |
October 1985 |
Knudson et al. |
4645744 |
February 1987 |
Charlton et al. |
4762799 |
August 1988 |
Seitz et al. |
5354825 |
October 1994 |
Klainer et al. |
5405975 |
April 1995 |
Kuhn et al. |
5837446 |
November 1998 |
Cozzette et al. |
5837454 |
November 1998 |
Cozzette et al. |
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Foreign Patent Documents
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2119840 |
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Sep 1994 |
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CA |
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0207392a3 |
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Jan 1987 |
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EP |
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0484865 |
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May 1992 |
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EP |
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0582836 |
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Feb 1994 |
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EP |
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0623599 |
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Nov 1994 |
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EP |
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9117432 |
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Nov 1991 |
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WO |
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9210739 |
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Jun 1992 |
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WO |
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Other References
Analytical Chemistry, vol. 62, pp. 2054-2055 (1990). .
Chem. Abstract 72:122891Y. .
Chemical, Biochemical and Environmental Fiber Sensors II, vol.
1368, pp. 165-174 (1990). .
He, H. et al. Novel type of ion-selective fluorosensor based on the
inner filter effect: an optrode for potassium, Anal. Chem., vol.
65, pp. 123-127, (1993). .
Journal of Fluorescence, vol. 2(2), pp. 93-97 (Jun. 1992). .
Morf et al., Pure & Appl. Chem., vol. 61, pp. 1613-1618 (1989).
.
Roe et al., Analyst, vol. 115, pp. 353-358 (1990). .
The Analyst, vol. 113(5), pp. 693-697 (May 1988). .
Wang et al., Analytical Science, vol. 6, pp. 715-720
(1990)..
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Primary Examiner: Snay; Jeffrey
Attorney, Agent or Firm: Wenderoth, Lind & Ponack,
L.L.P.
Parent Case Text
This application is a continuation of U.S. application Ser. No.
08/704,703, filed Nov. 15, 1996, now abandoned, the entire contents
of which are incorporated herein by reference, which was the
National Stage of International Application No. PCT/IB95/00159,
filed Mar. 13, 1995.
Claims
What is claimed is:
1. A composition comprising
(a) a transparent support
(b) which is coated on at least one side with a transparent coating
which comprises
(b1) a plasticizer-free, hydrophobic polymer having a glass
transition temperature T.sub.g of from -150 to 50.degree. C.,
(b2) counterions in the form of lipophilic salts,
(b3) an ionophore which forms a complex with the ion to be
determined, and
(b4) a compound of the formula I or II as fluorophore ##STR10## in
which R.sub.1 and R.sub.3, and R.sub.4 and R.sub.6 are C.sub.1
-C.sub.30 alkyl or C.sub.1 -C.sub.30 alkyl-CO--, and R.sub.2 and
R.sub.5 are H or C.sub.1 -C.sub.30 alkyl, with the proviso that the
total number of carbon atoms in the alkyl groups is at least 5, or
a salt thereof with an inorganic or organic acid,
wherein components (b1) to (b4) are not covalently bonded to one
another.
2. A composition according to claim 1, wherein R.sub.2 is H.
3. A composition according to claim 1, wherein the alkyl groups are
linear alkyl groups.
4. A composition according to claim 1, wherein the alkyl groups
contain 1 to 22 carbon atoms.
5. A composition according to claim 1, wherein R.sub.1 and R.sub.3
are C.sub.6 -C.sub.24 alkyl or C.sub.6 -C.sub.24 alkyl-CO--, and
R.sub.2 is H.
6. A composition according to claim 5, wherein R.sub.1 and R.sub.3
are C.sub.10 -C.sub.24 alkyl or C.sub.10 -C.sub.24 alkyl-CO--.
7. A composition according to claim 5, wherein R.sub.1 and R.sub.3
are C.sub.14 -C.sub.22 alkyl or C.sub.14 -C.sub.22 alkyl-CO--.
8. A composition according to claim 1, wherein R.sub.5 is H and
R.sub.4 and R.sub.6 are C.sub.6 -C.sub.24 alkyl.
9. A composition according to claim 8, wherein R.sub.4 and R.sub.6
are C.sub.10 -C.sub.24 alkyl.
10. A composition according to claim 9, wherein R.sub.4 and R.sub.6
are C.sub.14 -C.sub.22 alkyl.
11. A composition according to claim 1, wherein R.sub.4 and R.sub.5
are C.sub.1 -C.sub.6 alkyl, and R.sub.6 is C.sub.10 -C.sub.24 alkyl
or C.sub.10 -C.sub.22 alkyl-CO--.
12. A composition according to claim 11, wherein R.sub.4 and
R.sub.5 are C.sub.1 -C.sub.4 alkyl, and R.sub.6 is C.sub.14
-C.sub.22 alkyl or C.sub.14 -C.sub.22 alkyl-CO--.
13. A composition according to claim 12, wherein R.sub.4 and
R.sub.5 are methyl or ethyl, and R.sub.6 is C.sub.16 -C.sub.22
alkyl or C.sub.16 -C.sub.22 alkyl-CO--.
14. A composition according to claim 1, wherein the salt of the
compound of the formula I or II is derived from HF, HCl, HBr, HI,
H.sub.2 SO.sub.3, H.sub.2 SO.sub.4, H.sub.3 PO.sub.3, H.sub.3
PO.sub.4, HNO.sub.2, HNO.sub.3, HCIO.sub.4, HBF.sub.4, HPF6,
HSbF.sub.6, CF.sub.3 SO.sub.3 H, HB[C.sub.6 H.sub.5 ].sub.4,
toluenesulfonic acid, C.sub.1 -C.sub.4 alkyl- or phenylphosphonic
acid, formic acid, acetic acid, propionic acid, benzoic acid,
mono-, di- or trichloroacetic acid, or mono-, di- or
trifluoroacetic acid.
15. A composition according to claim 14, wherein the salt of the
compound of the formula I or II is derived from HCl, HBr, H.sub.2
SO.sub.4, HClO.sub.4, HBP.sub.4, HPF.sub.6, HB[C.sub.6 H.sub.5
].sub.4 or HSbF.sub.6.
16. A composition according to claim 1, wherein the compound of the
formula I or II has a pK.sub.a value of at least 8.
17. A composition according to claim 16, wherein the pK.sub.a value
is at least 10.
18. A composition according to claim 1, wherein the support is a
glass.
19. A composition according to claim 1, wherein the thickness of
the coating on the support is from 0.01 to 100 .mu.m.
20. A composition according to claim 1, wherein the hydrophobic
polymer has a molecular weight of at least 5 000 daltons.
21. A composition according to claim 1, wherein the hydrophobic
polymer is selected from the group consisting of the polyolefins,
polyesters, polyamides, polyethers, polyimides, polyesteramides,
polyamideimides, polyurethanes, polyetherurethanes,
polyesterurethanes, polyureas, polyurethaneureas and
polysiloxanes.
22. A composition according to claim 21, wherein the polymers
contain ionizable basic or acidic groups.
23. A composition according to claim 21, wherein the hydrophobic
polymers are polyurethanes made from polyethers of C.sub.3 -C.sub.6
alkanediols and aliphatic, cycloaliphatic,
cycloaliphatic-aliphatic, aromatic-aliphatic or aromatic C.sub.2
-C.sub.20 diisocyanates.
24. A composition according to claim 21, wherein the hydrophobic
polymers are copolymers comprising
a) from 10 to 90 mol % of identical or different structural units
of the formula III ##STR11## and from 90 to 10 mol % based on the
polymer, of identical or different structural units of the formula
IV, ##STR12## in which R.sub.7 and R.sub.8, independently of one
another, are H or C.sub.1 -C.sub.4 alkyl, X is --O-- or --NR.sub.14
--, R.sub.9 is C.sub.6 -C.sub.20 alkyl and R.sub.14 is H or C.sub.1
-C.sub.20 alkyl; R.sub.10 and R.sub.11, independently of one
another, are H, F, Cl or C.sub.1 -C.sub.4 alkyl, R.sub.12 and
R.sub.13, independently of one another, are H, F, Cl, C.sub.1
-C.sub.4 alkyl, --COOH, --COO--C.sub.1 -C.sub.5 alkyl,
--CONHC.sub.1 -C.sub.5 alkyl or --CON(R.sub.14)C.sub.1 -C.sub.5
alkyl, or R.sub.12 is H and R.sub.13 is --CN, phenyl, chlorophenyl,
C.sub.1 -C.sub.12 alkoxy or C.sub.2 -C.sub.18 acyloxy.
25. A composition according to claim 1, wherein the salt with a
lipophilic anion is an alkali metal, alkaline earth metal or
ammonium salt with a substituted or unsubstituted
tetraphenylborate.
26. A composition according to claim 25, wherein the cation is
Li.sup..sym., Na.sub..sym., K.sup..sym., Mg.sup.2.sym.,
Ca.sup.2.sym., NH.sub.4.sup..sym. or an ammonium cation of a
primary, secondary or tertiary amine or a quaternary ammonium
cation containing 1 to 40 carbon atoms.
27. A composition according to claim 25, wherein the borate anion
is tetraphenylborate, whose phenyl groups are unsubstituted or
substituted by one or more C.sub.1 -C.sub.4 alkyl, C.sub.1 -C.sub.4
alkoxy, halogen or trifluoromethyl groups.
28. A composition according to claim 25, wherein the borate anion
is tetraphenylborate, tetra(3,5-bistrifluoromethylphenyl)borate or
tetra(4-chlorophenyl)borate.
29. A composition according to claim 16, wherein the amount of the
salt with a lipophilic anion is from 0.01 to 10% by weight, based
on the amount of polymer.
30. A composition according to claim 1, wherein the polymer coating
contains an ionophore in an amount of from 0.01 to 10% by weight,
based on the amount of polymer.
31. A composition according to claim 1, wherein the potassium
ionophore is valinomycin.
32. A composition according to claim 1, wherein the amount of the
compound of the formula I or II is from 0.01 to 10% by weight,
based on the amount of polymer.
33. A composition according to claim 32, wherein the amount of the
compound of the formula I or II is from 0.1 to 5% by weight.
34. A composition according to claim 32, wherein the amount of the
compound of the formula I or II is from 0.1 to 2% by weight.
35. A composition according to claim 1, wherein the total number of
carbon atoms in the alkyl groups is at least 10.
36. A composition according to claim 1, wherein the total number of
carbon atoms in the alkyl groups is at least 12.
37. The composition as claimed in claim 1, wherein the composition
is formed by mixing the polymer, counterions, ionophore, and
fluorophore together to form a coating, and applying the coating to
the transparent support.
38. The composition as claimed in claim 37, wherein the polymer,
counterions, ionophore, and fluorophore are mixed together in a
single step.
39. A composition comprising
(a) a plasticizer-free, hydrophobic polymer having a glass
transition temperature Tg of from -150 to 50.degree. C., and
(b) a compound of the formula I or II as fluorophore ##STR13## in
which R.sub.1 and R.sub.3, and R.sub.4 and R.sub.6 are C.sub.1
-C.sub.30 alkyl or C.sub.1 -C.sub.30 alkyl-CO--, and R.sub.2 and
R.sub.5 are H or C.sub.1 -C.sub.30 alkyl, with the proviso that the
total number of carbon atoms in the alkyl groups is at least 5, or
a salt thereof with an inorganic or organic acid,
(c) an ionophore which forms a complex with the ion to be
determined, and
(d) counterions in the form of lipophilic salts,
wherein components (a) to (d) are not covalently bonded to one
another.
40. The composition as claimed in claim 39, wherein the composition
is formed by mixing the polymer, counterions, ionophore, and
fluorophore together.
41. The composition as claimed in claim 40, wherein the polymer,
counterions, ionophore, and fluorophore are mixed together in a
single step.
42. An optical sensor for the determination of ions in aqueous
measurement samples, in particular by means of fluorescence
spectrometry, which comprises
(a) a transparent support
(b) which is coated on at least one side with a transparent coating
which comprises
(b1) a plasticizer-free, hydrophobic polymer having a glass
transition temperature T.sub.g of from -150 to 50.degree. C.,
(b2) the salt of a lipophilic anion,
(b3) an ionophore which forms a complex with the ion to be
determined, and
(b4) a compound of the formula I or II as fluorophore ##STR14## in
which R.sub.1 and R.sub.3, and R.sub.4 and R.sub.6 are C.sub.1
-C.sub.30 alkyl or C.sub.1 -C.sub.30 alkyl-CO--, and R.sub.2 and
R.sub.5 are H or C.sub.1 -C.sub.30 alkyl, with the proviso that the
total number of carbon atoms in the alkyl groups is at least 5, or
a salt thereof with an inorganic or organic acid,
wherein components (b1) to (b4) are not covalently bonded to one
another.
43. A method for the optical determination of ions in aqueous
measurement samples, in which a sensor according to claim 42 is
brought into contact with said aqueous measurement sample, and the
change in fluorescence of the fluorophore in the active polymer
coating is then measured.
Description
The invention relates to a sensor for the optical determination of
ions, for example cations from the group consisting of metal and
ammonium cations, or for example anions from the group consisting
of anions of inorganic and organic acids, in aqueous samples by the
fluorescence method, which sensor contains certain highly basic
dyes from the group consisting of rhodamines and acridines as
fluorophores in the active coating, to a process for the
qualitative or quantitative determination of cations, in particular
in aqueous solutions, using the optical sensor, and to a
composition containing the fluorophores and polymers.
The optical determination of ions has recently achieved increased
importance, the presence or concentration of ions being measured,
for example, via the change in absorption or fluorescence of a
suitable dye. The sensors, also known as optrodes, generally
comprise a transparent support material and an active coating. This
active coating generally contains a transparent hydrophobic polymer
and a lipophilic plasticizer for achieving adequate ion diffusion
and solubility of the active constituents. The active constituents
are a specific ionophore as complexing agent for ions, a counterion
for maintaining electrical neutrality, and an indicator substance
which, due to a chemical change or a physical change in the
environment, generates a measurable optical signal.
U.S. Pat. No. 4,645,744 describes systems of this type in which the
indicator substance is a neutral compound, for example a dye
(p-nitrophenol), which interacts with an ionophore/metal cation
complex, causing a colour change as the optically measurable
signal. The interaction can cause, for example, the elimination of
a proton from the dye, causing a change in the electron state.
Suitable compounds include fluorescing compounds (for example
fluorescein), whose fluorescence changes due to the change in the
electron state and can be determined optically by means of
fluorescence measurements.
H. He et al. in Chemical, Biochemical and Environmental Fiber
Sensors II, SPIE Vol. 1368, pages 165 to 174 (1990), describe
systems containing a proton carrier (Nile Blue) as indicator
substance, in which the transport of potassium into the active
coating by means of valinomycin as ionophore causes dissociation of
the proton carrier and diffusion of a proton into the aqueous
phase. The proton carrier changes its colour from blue to red and,
depending on the choice of wavelength, either the reduction in
fluorescence of the blue dye or the corresponding increase in the
fluorescence of the red dye can be determined. Due to the higher
sensitivity and selectivity, measurement of the fluorescence is
preferred. A significant disadvantage of the process is the low
sensitivity of the system, due to the low fluorescence quantum
yield of the indicator dye used.
J. N. Roe in Analyst, Vol. 115, pages 353 to 358 (1990), describes
a system based on energy transfer due to complex formation of the
fluorescence dye used with the anionic form of a certain
indonaphthol, which itself forms a ternary complex with the
potassium-charged ionophore. The potassium is determined by
measuring the change in absorption after charging with potassium or
from the change in fluorescence. The sensitivity and response
speeds of this system are regarded as unsatisfactory.
Y. Kawabata in Anal. Chem. Vol. 62, pages 1528-1531 and 2054 to
2055, describes a membrane system for the optical determination of
potassium using a hydrophobic ion exchanger, namely
3,6-bis(dimethylamino)-10-dodecyl- or -10-hexadecylacridinium
bromide. A change in fluorescence is achieved by changing the
polarity in the microenvironment of the sample, since the
acridinium salts diffuse at the interface with the aqueous phase
due to ion exchange with the potassium ion.
W. E. Morf et al. in Pure & Appl. Chem., Vol. 61, No. 9, pages
1613 to 1618 (1989), describe the use of pH-sensitive chromo- or
fluoroionophores for the optical determination of cations based on
ion exchange reactions. The sensitivity of these systems is
relatively low, the measurement is hindered in optically dense
systems, and relatively high concentrations of chromo- or
fluoroionophores in the membrane are required.
K. Wang et al. in Analytical Science, Vol. 6, pages 715 to 720
(1990), describe membranes containing an absorption dye (Nile Blue)
as indicator substance for the optical measurement of metal
cations. The system is based on an ion exchange mechanism which
reduces the absorption by protonation of the dye. The sensitivity
of the system is regarded as too low.
Hitherto, no systems have been disclosed which have an ion exchange
mechanism for the optical measurement of ions and are based on the
determination of the change in fluorescence of a fluorophore and
have high sensitivity, since the fluorescence quantum yields and
basicities of the known pH-sensitive fluorophores are too low.
The systems disclosed hitherto contain high-molecular-weight,
hydrophobic polymers in combination with a plasticizer in the
active coating in order to ensure rapid response times and adequate
sensitivities. In these membrane materials, the long-term stability
and repeated use are considerably restricted, since the plasticizer
and other low-molecular-weight constituents, for example ionophores
or fluorophores, are washed out in the course of time. Furthermore,
low-molecular-weight substances can penetrate into the membrane and
render the sensor unusable.
It has now been found that certain acridine dyes and rhodamine dyes
surprisingly satisfy these high requirements and are lipophilic,
pH-sensitive and highly basic fluorophores which are highly
suitable, in a neutral polymer membrane together with an ionophore
and a counterion, for the determination of ions by the ion exchange
mechanism and have a fluorescence which is highly dependent on the
corresponding ion concentrations. These fluorophores are
distinguished by a high fluorescence quantum yield, high basicity,
a large difference between the fluorescence signals of the
protonated and deprotonated forms, high lipophilicity, adequate
photostability and suitable absorption and emission wavelengths.
Highly sensitive systems for the optical determination of ions on
the basis of fluorescence measurements can be provided.
Furthermore, it has been found, surprisingly, that the service life
and use frequency can be considerably increased, since
plasticizer-free, hydrophobic polymers having a defined glass
transition range can be employed as the polymers in the
membrane.
The invention relates to a composition comprising
(a) a transparent support
(b) which is coated on at least one side with a transparent coating
which comprises
(b1) a plasticizer-free, hydrophobic polymer having a glass
transition temperature Tg of from -150 to 50.degree. C.,
(b2) counterions in the form of lipophilic salts,
(b3) an ionophore which forms a complex with the ion to be
determined, and
(b4) a compound of the formula I or II as fluorophore ##STR2## in
which R.sub.1 and R.sub.3, and R.sub.4 and R.sub.6 are C.sub.1
-C.sub.30 alkyl or C.sub.1 -C.sub.30 alkyl-CO--, and R.sub.2 and
R.sub.5 are H or C.sub.1 -C.sub.30 alkyl, with the proviso that the
total number of carbon atoms in the alkyl groups is at least 5, and
salts thereof with inorganic or organic acids.
The total number of carbon atoms in the alkyl groups is preferably
at least 8, particularly preferably at least 10, especially
preferably at least 12.
In a preferred embodiment, R.sub.2 is H.
The alkyl groups can be linear or branched and preferably contain 1
to 22 carbon atoms. Linear alkyl groups are preferred. Examples of
alkyl are methyl, ethyl and the isomers of propyl, butyl, pentyl,
hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,
tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl,
nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl and
tricontyl. In a preferred embodiment, R.sub.1 and R.sub.3 are
C.sub.6 -C.sub.24 alkyl or C.sub.6 -C.sub.24 alkyl-CO--,
particularly preferably C.sub.10 -C.sub.24 alkyl or C.sub.10
-C.sub.24 alkyl-CO--, especially preferably C.sub.14 -C.sub.22
alkyl or C.sub.14 -C.sub.22 alkyl-CO--, while R.sub.2 is H. In
another embodiment, R.sub.5 is preferably H, and R.sub.4 and
R.sub.6 are preferably C.sub.6 -C.sub.24 alkyl, particularly
preferably C.sub.10 -C.sub.24 alkyl, especially preferably C.sub.14
-C.sub.22 alkyl. In a further embodiment, R.sub.4 and R.sub.5 are
preferably C.sub.1 -C.sub.6 alkyl, particularly preferably C.sub.1
-C.sub.4 alkyl, especially preferably methyl or ethyl, and R.sub.6
is C.sub.10 -C.sub.24 alkyl or C.sub.10 -C.sub.24 alkyl-CO--,
preferably C.sub.14 -C.sub.22 alkyl or C.sub.14 -C.sub.22
alkyl-CO--, especially preferably C.sub.16 -C.sub.22 alkyl or
C.sub.16 -C.sub.22 alkyl-CO--.
The salts of the compounds of the formulae I and II can be derived,
for example, from HF, HCl, HBr, HI, H.sub.2 SO.sub.3, H.sub.2
SO.sub.4, H.sub.3 PO.sub.3, H.sub.3 PO.sub.4, HNO.sub.2, HNO.sub.3,
HClO.sub.4, HBF.sub.4, HB(C.sub.6 H.sub.5).sub.4, HPF.sub.6,
HSbF.sub.6, CF.sub.3 SO.sub.3 H, toluenesulfonic acid, C.sub.1
-C.sub.4 alkyl- or phenylphosphonic acid, formic acid, acetic acid,
propionic acid, benzoic acid, mono-, di- or trichloroacetic acid,
or mono-, di- or trifluoroacetic acid. Preference is given to HCl,
HBr, H.sub.2 SO.sub.4, HClO.sub.4, HBF.sub.4, HB(C.sub.6
H.sub.5).sub.4, HPF.sub.6 and HSbF.sub.6.
The compounds of the formulae I and II are novel and can be
prepared in a manner known per se from commercial
3,6-diaminoacridine by stepwise alkylation by means of various
alkylating agents or alkylation using one alkylating agent or
acylating agent. Examples of suitable alkylating agents are dialkyl
sulfates or monohaloalkanes, in particular chloro-, bromo- and
iodoalkanes. Examples of suitable acylating agents are carboxylic
anhydrides and in particular carboxylic acid halides, for example
carboxylic acid chlorides. This reaction can be carried out in the
presence of inert polar and aprotic solvents, for example ethers,
alkylated acid amides and lactams or sulfones, and at elevated
temperatures, for example from 50 to 150.degree. C. It is expedient
to add a hydrogen halide scavenger, for example alkali metal
carbonates or tertiary amines, in particular sterically hindered
tertiary amines.
The compounds of the formula II can be obtained, for example, by
reacting phthalic anhydride with 2 mol equivalents of
3-monoalkylaminophenol. Another possible preparation comprises
reacting 3-monoalkylaminophenol with 1 mol equivalent of
2-hydroxy-4-dialkylamino-2'-carboxybenzophenone. These reactions
are described, for example, in U.S. Pat. No. 4,622,400. The
reaction is expediently carried out in an inert solvent, for
example hydrocarbons or ethers. Molar amounts of a condensation
agent, for example Lewis acids, concentrated sulfuric acid,
perchloric acid or phosphoric acid, are advantageously added. The
reaction temperatures can be, for example, from 50 to 250.degree.
C.
The compounds of the formula I can be isolated in a conventional
manner by precipitation, crystallization or extraction and
purified, if necessary, by recrystallization or chromatography.
They are crystalline, red, red-brown or red-violet compounds.
The compounds of the formulae I and II are highly suitable as
fluorophoric dye indicators for the optical determination of ions
in an aqueous environment, in particular by measurement of the
change in fluorescence.
The compounds of the formulae I and II preferably have a pK.sub.a
value of at least 8, particularly preferably at least 10.
The support can be formed, for example, from a plastic material,
for example polycarbonate or acrylic sheeting, mineral materials or
glass and can have any desired shape, for example plates,
cylinders, tubes, tapes or fibres. Glasses are preferred.
The thickness of the coating on the support can be, for example,
from 0.01 to 100 .mu.m, preferably from 0.1 to 50 .mu.m, more
preferably from 0.1 to 30 .mu.m, and particularly preferably from
0.1 to 10 .mu.m.
Various types of hydrophobic polymer are suitable for the
composition, where the term hydrophobic indicates that the water
content in the polymers is at most 15% by weight, preferably at
most 10% by weight, particularly preferably at most 5% by weight,
especially preferably at most 3% by weight, based on the polymer.
They expediently have a mean molecular weight of at least 5 000,
preferably at least 10 000 and particularly preferably at least 20
000 daltons, for example from 20 000 to 200 000 daltons, preferably
from 50 000 to 200 000 daltons. The polymers must have adequate
solubility in organic solvents so that they can be mixed with the
other components and can be converted into coatings by conventional
coating methods. They must furthermore be permeable to ions. The
glass transition temperature is preferably from -130 to 0.degree.
C. The dielectric constant of the polymers is preferably from 2 to
25, particularly preferably from 5 to 15, at 100 Hz and room
temperature. The optical transparency is preferably in the range
from 400 to 1200 nm, particularly preferably from 400 to 900
nm.
Suitable polymers are known to the person skilled in the art. They
can be homopolymers, copolymers, block polymers, graft polymers and
polymer alloys. The components of a polymer alloy may be a
combination of two or more polymer components, said components
having high and low glass transition temperatures. The glass
transition temperature can be adjusted, for example, by means of
the polarity and the chain length and content of structural units.
The polymers can be selected, for example, from the group
consisting of polyolefins, polyesters, polyamides, polyethers,
polyimides, polyesteramides, polyamideimides, polyurethanes,
polyetherurethanes, polyesterurethanes, polyureas,
polyurethaneureas and polysiloxanes, it being possible for the
polymers to contain ionizable, basic groups (for example amino
groups) or ionizable, acidic groups (for example carboxyl or
sulfonyl groups), which may be used as replacement for a counterion
of lipophilic salts and can provide improved ion transport.
Some examples of monomers for the preparation of polyolefins are
C.sub.2 -C.sub.12 olefins, acrylic acid, methacrylic acid, maleic
acid, maleic anhydride, C.sub.1 -C.sub.30 esters of acrylic and
methacrylic acid, C.sub.1 -C.sub.30 amides of acrylic and
methacrylic acid, acrylamide and methacrylamide, vinyl esters of
C.sub.1 -C.sub.20 carboxylic acids, acrylonitrile, butadiene,
isoprene, chlorobutadiene, styrene, .alpha.-methylstyrene, vinyl
chloride, vinyl fluoride, vinylidene chloride and vinyl ethers of
C.sub.1 -C.sub.30 alcohols.
Polyesters, polyesteramides and polyamides are preferably
synthesized from C.sub.2 -C.sub.12 dicarboxylic acids and C.sub.2
-C.sub.18 diols or -diamines. Polyimides are preferably synthesized
from C.sub.2 -C.sub.18 tetracarboxylic acids and C.sub.2 -C.sub.18
diamines. Polyethers are preferably synthesized from aliphatic
C.sub.2 -C.sub.12 diols (1,2- or .alpha.,.omega.-lining) or linear
adducts of these diols and C.sub.8 -C.sub.30 diglycidyl ethers.
Polyurethanes and polyureas are preferably synthesized from C.sub.2
-C.sub.18 diols or -diamines and C.sub.2 -C.sub.20 diisocyanates
and/or triisocyanates. Polysiloxanes are preferably synthesized
from di(C.sub.1 -C.sub.4)alkylsilyldichlorosilanes.
In a preferred embodiment, the hydrophobic polymers are
polyurethanes made from polyethers of C.sub.3 -C.sub.6 alkanediols
and aliphatic, cycloaliphatic, cycloaliphatic-aliphatic,
aromatic-aliphatic or aromatic C.sub.2 -C.sub.20 diisocyanates, for
example from polytetrahydrofuran and
bis(p-diisocyanatocyclohexyl)methane (Tecoflex.RTM.).
In another preferred embodiment, the hydrophobic polymers are
copolymers comprising
a) from 10 to 90 mol %, preferably from 20 to 80 mol %,
particularly preferably from 30 to 70 mol %, of identical or
different structural units of the formula III ##STR3## and from 90
to 10 mol %, preferably from 80 to 20 mol %, particularly
preferably from 70 to 30 mol %, based on the polymer, of identical
or different structural units of the formula IV, ##STR4## in which
R.sub.7 and R.sub.8, independently of one another, are H or C.sub.1
-C.sub.4 alkyl, X is --O-- or --NR.sub.14 --, R.sub.9 is C.sub.6
-C.sub.20 alkyl and R.sub.14 is H or C.sub.1 -C.sub.20 alkyl;
R.sub.10 and R.sub.11, independently of one another, are H, F, Cl
or C.sub.1 -C.sub.4 alkyl, R.sub.12 and R.sub.13, independently of
one another, are H, F, Cl, C.sub.1 -C.sub.4 alkyl, --COOH,
--COO--C.sub.1 -C.sub.5 alkyl, --CONHC.sub.1 -C.sub.5 alkyl or
--CON(R.sub.14)C.sub.1 -C.sub.5 alkyl, or R.sub.12 is H and
R.sub.13 is --CN, phenyl, chlorophenyl, C.sub.1 -C.sub.12 alkoxy or
C.sub.2 -C.sub.18 acyloxy.
R.sub.7 is preferably H or methyl, and R.sub.8 is preferably H. X
is preferably --O--. R.sub.9 is preferably C.sub.6 -C.sub.18 alkyl.
Examples of R.sub.9 are hexyl, heptyl, octyl, 2-ethylhexyl, nonyl,
decyl, dodecyl, tetradecyl, hexadecyl and octadecyl.
R.sub.10 is preferably H or methyl, R.sub.11 is preferably H, and
R.sub.12 is preferably H. R.sub.13 is preferably --CN, phenyl,
--COO--C.sub.1 -C.sub.4 alkyl, C.sub.1 -C.sub.4 alkoxy or C.sub.2
-C.sub.6 acyloxy. Some examples of acyloxy are acetyloxy,
propionyloxy, butyroyloxy, pentanoyloxy and hexanoyloxy.
Examples of suitable salts with lipophilic anions are alkali metal,
alkaline earth metal and ammonium salts with substituted or
unsubstituted tetraphenylborates. Preferred cations are
Li.sup..sym., Na.sup..sym., K.sup..sym., Mg.sup.2.sym.,
Ca.sup.2.sym., NH.sub.4.sup..sym., and the ammonium cations of
primary, secondary and tertiary amines and quaternary ammonium
cations which can contain from 1 to 60 carbon atoms. Some examples
of ammonium cations are methyl-, ethyl-, propyl-, butyl-, hexyl-,
octyl-, decyl-, dodecyl-, tetradecyl-, hexadecyl-, octadecyl-,
dimethyl-, diethyl-, dibutyl-, butylmethyl-, dioctyl-, didoceyl-,
dodecylmethyl-, trimethyl-, triethyl-, tripropyl-, tributyl-,
trioctyl-, tridodecyl-, dodecyldimethyl-, didodecylmethyl-,
tetramethyl-, tetraethyl-, tetrapropyl-, tetrabutyl-, tetrahexyl-,
tetraoctyl-, tetradecyl-, tetradodecyl-, dodecyltrimethyl-,
octyltrimethyl-, didodecyldimethyl-, tridodecylmethyl-,
tetradecyltrimethyl- and octadecyltrimethylammonium. Quaternary
ammonium salts are preferred, in particular those having 4 to 48,
preferably 4 to 24, carbon atoms. Other suitable salts with
lipophilic anions are alkali metal, alkaline earth metal and
ammonium salts of anionic surfactants, for example of C.sub.12
-C.sub.22 fatty acids or C.sub.12 -C.sub.22 alkylsulfonic acids,
C.sub.12 -C.sub.22 alkylphosphates, C.sub.4 -C.sub.18 alkylbenzoic
acids, C.sub.4 -C.sub.18 alkylphenylsulfonic acids or C.sub.4
-C.sub.18 alkylphenylphosphonic acids.
An example of a suitable borate anion is tetraphenylborate, whose
phenyl groups may be substituted by one or more, preferably 1 to 3,
particularly preferably 1 or 2, C.sub.1 -C.sub.4 alkyl, C.sub.1
-C.sub.4 alkoxy, halogen, for example F or Cl, or trifluoromethyl
groups. Some specific examples are tetraphenylborate,
tetra(3,5-bistrifluoromethylphenyl)borate and
tetra(4-chlorophenyl)borate. The salts with lipophilic anions serve
as negative charge compensation for the metal cations diffusing
into the active coating and to be measured therein in complexed
form.
The salts with lipophilic anions can also be salts of polymers
containing acidic or basic groups, for example polysulfonic acids
or polycarboxylic acids. These polymers can also be structural
units (randomly distributed structural units or block elements) of
the hydrophobic polymers.
The amount of salts with lipophilic anions can be, for example,
from 0.01 to 10% by weight, preferably from 0.1 to 5% by weight,
particularly preferably from 0.1 to 2% by weight, based on the
amount of polymer.
The polymer coating (also referred to as membrane) contains an
ionophore in, for example, an amount of from 0.01 to 10% by weight,
preferably from 0.1 to 5% by weight, particularly preferably from
0.1 to 2% by weight, based on the amount of polymer. Ionophores are
natural or synthetic organic compounds which contain a plurality
of, usually alternating, electron-rich heteroatoms, for example S,
N and in particular O, in linear or cyclic carbon chains and which
are capable of selectively complexing the ions to be measured. The
natural compounds are frequently macrocyclic compounds, for example
valinomycin, which is capable of selectively binding potassium
cations. Another example is nonactin. A large group of ionophores
comprises macrocyclic polyethers (crown ethers), which are capable
of complexing various metal cations, depending on the geometry and
size. Further examples of ionophores are coronandenes, kryptandenes
and calixarenes. Examples of open-chain ionophores are podandenes.
Such ionophores are described, for example, in U.S. Pat. No.
4,645,744.
Examples of ionophores for anions are open-chain or macrocyclic
polyamines (mono- and polycyclic compounds, usually in protonated
form as polycations or as quaternary (poly)ammonium salts);
open-chain or macrocyclic (mono- and polycyclic) polyamides;
open-chain or macrocyclic (cyclic) polypyridinium compounds;
calixarenes; cyclodextrins; cobyrinates and metal porphyrin
complexes; open-chain or macrocyclic metallocene compounds; mono-
and polydentate ligand systems containing, for example, B, Si, Al
or Sn as complexing ligand atoms.
The amount of compounds of the formulae I and II can be, for
example, from 0.01 to 10% by weight, preferably from 0.1 to 5% by
weight, particularly preferably from 0.1 to 2% by weight, based on
the amount of polymer.
The fluorophores to be used according to the invention have very
suitable absorption and emission wavelength ranges which allow the
use of known and inexpensive light sources, for example halogen or
xenon lamps or light-emitting diodes. Examples of detectors which
can be employed are photodiodes. The fluorophores furthermore have
high absorption coefficients and high quantum yields can be
achieved. The high lipophilicity, high basicity and the large
dynamic range of the change in fluorescence between the protonated
and deprotonated forms satisfy, in particular, the high
requirements for optical determination of ions based on
fluorescence measurements. Both cations and anions can be
determined.
Examples of suitable cations are cations of metals from the first
to fifth main groups and the first to eighth sub-groups of the
Periodic Table of the Elements, the lanthanides and actinides. Some
examples of metals are Li, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, B, Al,
Ga, In, Tl, Sn, Pb, Sb, Bi, Cu, Ag, Au, Zn, Cd, Hg, Sc, Y, Ti, Zr,
Hf, Cr, Mo, W, Mn, Fe, Co, Ni, Ru, Os, Rh, Ir, Pt, Pd, La, Ce, Pr,
Nd, Pm, Sm, Eu, Gd, Th, Dy, Ho, Er, Yb, Lu, Ac, Th, Pa, U, Np and
Pu. Preferred cations are the alkali and alkaline earth metal ions,
in particular Li.sup..sym., Na.sup..sym., K.sup..sym.,
Mg.sup.2.sym., Ca.sup.2.sym. and Sr.sup.2.sym., very particularly
K.sup..sym., Na.sup..sym. and Ca.sup..sym.. Examples of suitable
ammonium cations are NH.sub.4.sup..sym. and the cations of
protonated primary, secondary and tertiary amines and quaternary
ammonium. The amines can contain from 1 to 40, preferably from 1 to
20, particularly preferably from 1 to 12, carbon atoms. The
quaternary ammonium can contain from 4 to 40, preferably from 4 to
20, particularly preferably from 4 to 16, carbon atoms.
The anions to be measured can be derived from mineral acids, oxygen
acids and inorganic complex acids. Examples are the halides and
pseudohalides F.sup..crclbar., Cl.sup..crclbar., Br.sup..crclbar.,
I.sup..crclbar., N.sub.3.sup..crclbar., CN.sup..crclbar.,
OCN.sup..crclbar. and SCN.sup..crclbar. ; anions of inorganic
oxygen acids NO.sub.2.sup..crclbar., NO.sub.3.sup..sym.,
CO.sub.3.sup..crclbar., PO.sub.4.sup.3.crclbar.,
SO.sub.4.sup.2.crclbar., ClO.sub.4.sup..crclbar.,
MnO.sub.4.sup..crclbar. and ClO.sub.3.sup..crclbar. ; anions of
inorganic complex acids Fe(CN).sub.6.sup.4.crclbar. and
Co(CN).sub.6.sup.3.crclbar. ; anions of carboxylic acids, phenols
and nucleotide anions, such as adenosine phosphate.
The composition according to the invention is highly suitable as an
optical sensor for the quantitative determination of ions, in
particular cations, very particularly metal cations, for example
potassium cations, in an aqueous environment, preferably by means
of fluorescence spectrometry. The determinations can be carried out
quickly with high accuracy even for low concentrations (for example
in the micromolar range to the nanomolar range), since the
pH-dependent equilibria of the complexing reactions and of proton
exchange become established quickly and the fluorophores are
characterized by a high fluorescence quantum yield and sensitivity.
The analyses can be carried out, for example, directly in body
fluids (blood, urine, serum), natural water or waste water, where
it may be possible for any interfering cations to be specifically
bound or removed in advance. The composition according to the
invention is particularly suitable for the determination of
physiological amounts, for example for potassium in the range from
0.5 to 10 mmol, of cations in aqueous media. By means of the
anionic compounds of the formula II, which generally have pK values
of below 6, this property of fluorophore can be used for the
determination of anions, particularly halides, especially chloride,
by the novel detection method, since a pK range of below 6 and, for
example, up to about 4 is favourable for this detection.
In addition to the preferred method of fluorescence spectroscopy,
other optical measurement methods may also be used, for example
surface plasmoresonance spectroscopy, absorption spectroscopy,
reflection spectroscopy, interferometry or surface-amplified Raman
or fluorescence spectroscopy.
The invention furthermore relates to a composition comprising
(a) a plasticizer-free, hydrophobic polymer having a glass
transition temperature Tg of from -150 to 50.degree. C. and
(b) a compound of the formula I or II as fluorophore ##STR5## in
which R.sub.1 and R.sub.3, and R.sub.4 and R.sub.6 are C.sub.1
-C.sub.30 alkyl or C.sub.1 -C.sub.30 alkyl-CO--, and R.sub.2 and
R.sub.5 are H or C.sub.1 -C.sub.30 alkyl, with the proviso that the
total number of carbon atoms in the alkyl groups is at least 5, or
salts thereof with inorganic or organic acids,
(c) an ionophore which forms a complex with the ion to be
determined, and
(d) counterions in the form of lipophilic salts.
The abovementioned preferences and embodiments apply to this
composition. The composition has a long shelf life and is a coating
composition for the production of sensors.
The novel composition may additionally comprise inert solvents,
where the concentration of the composition in the solution is from
1 to 50% by weight, preferably from 5 to 40% by weight,
particularly preferably from 5 to 30% by weight, based on the
solution.
Examples of suitable inert solvents are protic-polar and aprotic
solvents, which can be used alone or in mixtures of at least two
solvents. Examples are: ethers (dibutyl ether, tetrahydrofuran,
dioxane, ethylene glycol monomethyl and dimethyl ether, ethylene
glycol monoethyl and diethyl ether, diethylene glycol diethyl
ether, triethylene glycol dimethyl ether), halogenated hydrocarbons
(methylene chloride, chloroform, 1,2-dichloroethane,
1,1,1-trichloroethane, 1,1,2,2-tetrachloroethane), carboxylates and
lactones (ethyl acetate, methyl propionate, ethyl benzoate,
2-methoxyethyl acetate, .gamma.-butyrolactone,
.delta.-valerolactone, pivalolactone), carboxamides and lactams
(N,N-dimethylformamide, N,N-diethylformamide,
N,N-dimethylacetamide, tetramethylurea, hexamethylphosphoric
triamide, .gamma.-butyrolactam, .epsilon.-caprolactam,
N-methylpyrrolidone, N-acetylpyrrolidone, N-methylcaprolactam),
sulfoxides (dimethyl sulfoxide), sulfones (dimethyl sulfone,
diethyl sulfone, trimethylene sulfone, tetramethylene sulfone),
tertiary amines (N-methylpiperidine, N-methylmorpholine), aliphatic
and aromatic hydrocarbons, for example petroleum ether, pentane,
hexane, cyclohexane, methylcyclohexane, benzene or substituted
benzenes (chlorobenzene, o-dichlorobenzene, 1,2,4-trichlorobenzene,
nitrobenzene, toluene, xylene) and nitrites (acetonitrile,
propionitrile, benzonitrile, phenylacetonitrile). The choice of a
solvent depends essentially on the solubility of the individual
components in the novel composition, which, as a coating for a
sensor, should give a highly homogeneous mixture. Preferred
solvents are aprotic polar solvents.
The invention furthermore relates to an optical sensor for the
determination of cations in aqueous measurement samples, in
particular by means of fluorescence spectrometry, which
comprises
(a) a transparent support,
(b) which is coated on at least one side with a transparent coating
comprising
(b1) a plasticizer-free, hydrophobic polymer having a glass
transition temperature of from -150 to 50.degree. C.,
(b2) a counterion in the form of a salt of a lipophilic anion,
(b3) an ionophore which forms a complex with the ion to be
determined, and
(b4) a compound of the formula I or II as the fluorophore.
The abovementioned preferences and embodiments apply to the
sensor.
The novel sensor is produced by coating the support. Conventional
processes, for example brushing, knife coating, dipping, spraying,
pouring, curtain coating and spin coating, can be used for this
purpose. In order to improve adhesion, the support can be provided,
before the coating, with adhesion-promoting layers, for example by
treatment with alkyl chlorosilanes.
The invention furthermore relates to a method for the optical
determination of ions in aqueous measurement samples, in which a
composition according to the invention is brought into contact with
said aqueous measurement sample, and then, in particular, the
change in fluorescence of the fluorophore in the active polymer
coating is measured.
The process according to the invention can be carried out, for
example, by immobilizing the composition according to the invention
comprising support and active polymer coating in an optical cell in
which the active coating is brought into contact with the
measurement sample. The optical cell contains a window through
which the active coating can be excited by irradiation and the
emitted fluorescence radiation can be measured by means of a
spectrofluorometer. The wavelengths are adjusted so that the
absorption is at a maximum for the irradiation and the emission is
at a maximum for the fluorescence measurement. The intensity is
measured as a function of time. The measurement system can be
designed so that the measurement is carried out discontinuously or
continuously, for example by pumping the measurement solution
through the measurement cell. In order to determine unknown
concentrations of cations, the system can first be calibrated by
means of measurement samples of known concentration, and the
concentrations are plotted as a function of the fluorescence
intensity. It is expedient to add pH buffers to the measurement
sample, since the sensitivity of the measurement, and consequently
also the fluorescence intensity of the fluorophore, depends on the
pH of the measurement solution due to the pH-dependence of the
absorption spectrum. In another embodiment, this pH-dependency can,
however, also be determined and taken into account in the
calculation. The pH range of the measurement sample can be, for
example, from 4 to 8, more preferably from 6.5 to 7.5. Examples of
suitable buffers are citrate buffers and phosphate buffers. Further
buffer systems are described in U.S. Pat. No. 4,645,744, in
particular including those which are incorporated directly into the
active coating in order to avoid addition to the measurement
sample.
The examples below illustrate the invention in greater detail.
A) Preparation of fluorophores of the formulae I and II
EXAMPLE A1
Preparation of 3,6-bis(n-octylamino)acridine
6.33 g of anhydrous potassium carbonate are added to a solution of
2.5 g of 3,6-diaminoacridine hydrochloride and 3.55 ml of
1-bromooctane in 50 ml of dimethyl sulfoxide, and the mixture is
stirred at 80.degree. C. for 48 hours. The cooled reaction mixture
is subsequently poured onto ice, and the brown suspension is
extracted with methylene chloride. The organic phase is washed with
saturated aqueous NaCl solution and dried over sodium sulfate.
After evaporation, the red-brown oil is chromatographed on silica
gel using methylene chloride/methanol (9:1). After evaporation of
the solvent, the residue is taken up in diethyl ether/methanol
(10:1) and chromatographed on aluminium oxide. The eluate is taken
up in methanol, 2N HCl is added, the mixture is extracted with
diethyl ether, and the ether phase is dried and evaporated. The
residue is dissolved in methylene chloride, n-hexane is added, and
the red crystalline precipitate formed is filtered off. Further
product can be isolated from the mother liquor after evaporation.
The melting point of the title compound is 245.degree. C.
Absorption spectrum (ethanol): .lambda..sub.max =472 nm;
.epsilon.=51 400.
EXAMPLE A2
Preparation of 3,6-bis(n-eicosylamino)acridine
2.53 g of anhydrous potassium carbonate are added to a solution of
2.5 g of 3,6-diaminoacridine hydrochloride and 2.95 g of 1-eicosyl
bromide in 20 ml of N,N'-dimethylethyleneurea, and the mixture is
stirred at 50.degree. C. for 86 hours. The cooled reaction mixture
is subsequently poured into water, and the orange-brown suspension
is extracted with methylene chloride. The organic phase is washed
with water and dried over sodium sulfate. After evaporation, 2N HCl
is added to the brown oil. The red precipitate formed is filtered
off, washed with water and then dried in a high vacuum. The
resultant red-brown crystals are taken up in methylene
chloride/methanol (10:1) and chromatographed on silica gel. After
evaporation, the residue is taken up in diethyl ether/methanol
(10:1) and re-chromatographed on silica gel, giving the title
compound as red crystals, absorption spectrum (ethanol):
.lambda..sub.max =472 nm; .epsilon.=42 200.
EXAMPLE A3
Preparation of 3,6-bis(n-hexylamino)acridine
298 mg of ground potassium hydroxide are added to a solution of 500
mg of N,N'-bistosyl-3,6-diaminoacridine and 797 mg of 1-bromohexane
in 25 ml of dimethylformamide, and the mixture is stirred at
60.degree. C. for 22 hours. The cooled reaction mixture is
subsequently poured into water and extracted with ethyl acetate,
and the organic phase is separated off, washed with aqueous NaCl
solution and then dried over sodium sulfate. Evaporation gives a
dark-red oil, which is taken up in toluene/ethyl acetate (20:1) and
chromatographed on silica gel. Evaporation of the solvent gives a
yellow, viscous oil, which is dissolved in 11.5 ml of glacial
acetic acid, 4.6 ml of 97% sulfuric acid are added with water
cooling, and the mixture is then stirred at room temperature for 15
hours. The red reaction mixture is poured into ice water and
adjusted to pH 11 by means of 30% NaOH. The mixture is extracted
with ethyl acetate, and the organic phase is washed with 2N HCl and
saturated aqueous NaCl solution and then dried over sodium sulfate.
After evaporation, the dark-red, viscous oil is taken up in t-butyl
methyl ether/methanol (5:1) and chromatographed on silica gel,
giving the title compound as orange-red crystals having a melting
point of >200.degree. C. (decomposition). .sup.1 H-NMR
(CDCl.sub.3): 8.1 [s, 1H, C(9)]; 7.44 [d, 2H, C(8)]; 6.93 [s, 2H,
C(S)]; 6.82 [d, 2H, C(7)]; 3.20 [t, 4H, N-CH.sub.2 ]; 1.68 [m, 6H,
CH.sub.3 ].
EXAMPLE A4
Preparation of 3,6-bis(n-heptylcarbonylamino)acridine
3.1 ml of heptanoyl chloride are slowly added dropwise to a
suspension of 2.5 g of 3,6-diaminoacridine hydrochloride in 50 ml
of pyridine, and the mixture is then stirred for 30 minutes. The
reaction mixture is subsequently poured into water. The yellow
suspension is extracted with methylene chloride, and the organic
phase is washed with aqueous saturated NaCl solution and dried over
sodium sulfate. After evaporation, the dark-red oil is taken up in
methylene chloride/methanol (10:1) and chromatographed on silica
gel. The evaporated eluate is taken up in methylene chloride and
added dropwise to cyclohexane. The yellow precipitate formed is
filtered off, washed with cyclohexane and dried in a high vacuum,
giving the title compound as yellow crystals having a melting point
of 243-244.degree. C. Absorption spectrum (ethanol):
.lambda..sub.max =384 nm; .epsilon.=2 300.
EXAMPLE A5
Preparation of ##STR6##
a) A solution of 12.8 g of phthalic anhydride and 13.2 g of
3-N,N-diethylaminophenol is stirred at 110.degree. C. for 16 hours
in 75 ml of toluene. The precipitated product is filtered off and
recrystallized from ethanol, giving brick-red crystals of
1-carboxy-1'-hydroxy-3'-diethylaminobenzophenone (product A) having
a melting point of 214.degree. C.
b) A solution of 5.5 g of 3-aminophenol and 21.6 g of
1-bromoeicosane in 250 ml of 1,4-dioxane is stirred at 100.degree.
C. for 48 hours. The mixture is evaporated in vacuo, and the brown,
gelatinous residue is taken up in toluene/ethyl acetate (10:1) and
chromatographed on silica gel, giving 3-N-eicosylaminophenol as
white crystals having a melting point of 80.degree. C.
c) 626 mg of product A and 790 mg of 3-N-eicosylaminophenol are
stirred for 2 hours at 170.degree. C. in 5 ml of phosphoric acid
(85%). After cooling, a solution of 1 ml of concentrated HCl in 1
ml of methanol is added, and the mixture is subsequently extracted
with methylene chloride. After removal of the solvent, the residue
is taken up in methylene chloride/methanol (85:15) and
chromatographed on silica gel, giving the title compound as
red-violet crystals having a melting point of 115.degree. C.
Absorption spectrum (ethanol): .lambda..sub.max =532 nm;
.epsilon.=90 000.
EXAMPLE A6
Preparation of ##STR7##
a) A solution of 5.45 g of 3-aminophenol and 11.6 g of
1-bromooctane in 250 ml of dioxane is stirred at 100.degree. C. for
80 hours, the solvent is then evaporated, and the residue is then
taken up in toluene/ethyl acetate (10:1) and chromatographed on
silica gel, giving N-octylaminophenol as beige crystals, melting
point 75.degree. C.
b) 1.1 g of N-octylaminophenol and 0.37 g of phthalic anhydride are
melted together at 100.degree. C. 1 ml of phosphoric acid (85%) is
added to the melt, which is then heated to 170.degree. C. After 1
hour, the mixture is allowed to cool and 2N HCl is added. The
mixture is extracted with methylene chloride, the solvent is
removed, and the red residue is taken up in methylene
chloride/methanol (85:15). Chromatography on silica gel gives the
title compound as red crystals having a melting point of
183.degree. C. Absorption spectrum (ethanol): .lambda..sub.max =522
nm; .epsilon.=73 700.
EXAMPLE A7
Preparation of ##STR8##
a) 1.57 g of product A from Example A5a, 0.55 g of 3-aminophenol
and 10 ml of phosphoric acid (85%) are stirred for 30 minutes at
170.degree. C. 6.7 ml of perchloric acid (50%) and 100 ml of
methanol are then added, the mixture is re-heated, and the solvent
is then removed in vacuo. The residue is taken up in methylene
chloride, the solution is washed with water, and the solvent is
removed again. The residue is taken up in methylene
chloride/methanol (10:1) and chromatographed on silica gel, giving
red crystals of compound B of the formula ##STR9## having a melting
point of 175.degree. C.
b) 0.1 g of compound B is dissolved in 1 ml of methylene chloride
and 0.3 ml of pyridine, and 100 mg of stearoyl chloride are added.
After 3 hours, the mixture is evaporated to dryness in vacuo, and
the residue is dissolved in methylene chloride/methanol (85:15) and
chromatographed on silica gel, giving the title compound as red
crystals having a melting point of 145.degree. C. Absorption
spectrum (ethanol): .lambda..sub.max =560 nm; .epsilon.=10 900.
B) Preparation of polymers
EXAMPLES B1 TO B7
The monomers listed in Table 1 are introduced into an ampoule in
the stated mixing ratios together with 0.1% by weight of
.alpha.,.alpha.'-azobisisobutyronitrile. In order to remove oxygen,
the ampoule is evacuated and filled with nitrogen a number of
times, then sealed, warmed to 60.degree. C. and left at this
temperature for 48 hours. The mixture is then cooled and dissolved
in ten times the amount (based on the monomers) of tetrahydrofuran
(THF). This solution is transferred into 20 times the amount of
methanol, and the precipitated polymer is then filtered off. The
dried polymer is re-dissolved in THF and precipitated using
methanol, separated off and then dried in vacuo for 48 hours.
In Table 1 below, the following abbreviations are used:
AN=acrylonitrile, DodMA=dodecyl methacrylate, EHA=ethylhexyl
acrylate, MMA=methyl methacrylate, VAC=vinyl acetate. The inherent
viscosity (IV) is determined at 25.degree. C. in a solution of 0.5%
by weight of polymer in THF.
TABLE 1 ______________________________________ Ex- am- ple DodMA AN
VAC EHA MMA Yield IV No. (mol %) (mol %) (mol %) (mol %) (mol %) (%
by wt.) ______________________________________ (dl/g) B1 50 25 25
-- -- 76 1.765 B2 60 20 20 -- -- 50 0.229 B3 40 12 48 -- -- 66
1.075 B4 40 6 54 -- -- 52 1.092 B5 40 40 -- -- 20 87 2.560 B6 -- 40
20 40 -- 90 1.386 ______________________________________
EXAMPLES B6 AND B7
The procedure is as in Examples B1-B6, using ethyl acetate (EA),
acrylonitrile (AN) and ethylhexyl acrylate (EHA). The results are
shown in Table 2.
TABLE 2 ______________________________________ Example EA AN EHA
Yield IV No. (mol %) (mol %) (mol %) (% by wt.) (dl/g)
______________________________________ B7 90 10 -- 54 1.394 B8 --
10 90 82 0.663 ______________________________________
C) Production of coated supports
EXAMPLES C1-C8
a) Support material
The support material used is pretreated glass. Circular glass
sheets (diameter 18 mm, thickness 0.17 mm) are immersed for one
hour in a solution of 10% by volume of dimethyldodecylchlorosilane
in isopropanol. The glass sheets are then each washed one after the
other with 200 ml of isopropanol, ethanol and methanol and dried at
110.degree. C. for 1 hour. The hydrophobicized surface has better
adhesion of the membrane coating.
b) Preparation of the coating solution.
The following constituents are introduced into a 2 ml bottle
together with tetrahydrofuran (THF) and shaken until the components
have dissolved. The fluorophore used is the compound of Example
A5.
EXAMPLE C1
125 mg of polymer from Example B1, 1.0 mg of fluorophore, 1.5 mg of
valinomycin, 1.2 mg of potassium
tetrakis[3,5-(trifluoromethyl)phenyl]borate, 3 ml of THF.
EXAMPLE C2
100 mg of polymer from Example B2, 1.0 mg of fluorophore, 1.5 mg of
valinomycin, 1.2 mg of potassium
tetrakis[3,5-(trifluoromethyl)phenyl]borate, 2 ml of THF.
EXAMPLE C3
40 mg of polymer from Example B3, 1.5 mg of fluorophore, 1.5 mg of
valinomycin, 1.2 mg of potassium
tetrakis[3,5-(trifluoromethyl)phenyl]borate, 2 ml of THF.
EXAMPLE C4
38 mg of polymer from Example B4, 1.5 mg of fluorophore, 1.5 mg of
valinomycin, 1.2 mg of potassium
tetrakis[3,5-(trifluoromethyl)phenyl]borate, 2 ml of THF.
EXAMPLE C5
50 mg of polymer from Example B7, 1.5 mg of fluorophore, 1.5 mg of
valinomycin, 1.2 mg of potassium
tetrakis[3,5-(trifluoromethyl)phenyl]borate, 2 ml of THF.
EXAMPLE C6
20 mg of polyurethane Tecoflex.RTM. (Thermedics Inc., Woburn)
having a Tg of -70.degree. C., 0.5 mg of fluorophore, 0.24 mg of
valinomycin, 0.2 mg of potassium
tetrakis[3,5-(trifluoromethyl)phenyl]borate, 1 ml of THF.
EXAMPLE C7
100 mg of polyurethane Tecoflex.RTM. (Thermedics Inc., Woburn)
having a Tg of -70.degree. C., 3 mg of fluorophore, 50 mg of
diethyl
N,N-[(4R,5R)-4,5-dimethyl-1,8-dioxo-3,6-dioxaoctamethylene)bis(12-methylam
ino)dodecanoate (calcium ionophore, Fluka 21102), 6 mg of potassium
tetrakis[3,5-(trifuoromethyl)phenyl]borate, 2 ml of THF.
c) Production of coated glass supports.
The glass supports are clamped in the chamber of a spin-coating
apparatus (Optocoat OS 35var, Willer Company, CH-8484 Weisslingen).
The chamber is rinsed with 10 ml of tetrahydrofuran and rotated for
2 minutes at 3 800 revolutions/minute. 50 .mu.l of the particular
coating solution are then pipetted onto the glass support, and the
glass support is rotated for a further 10 seconds. The glass
support coated with a membrane is then removed and dried for 10
minutes in air.
D) Determination of ion concentrations
EXAMPLES D1 TO D6
The coated glass supports are clamped in an optical cell in which
the membrane is in contact with the measurement liquid. The
membrane can be optically excited in the optical cell and the
fluorescence radiation measured. The optical cell is introduced
into a spectrofluorometer (Perkin-Elmer LS-50). The absorption and
emission wavelengths are adjusted to the corresponding maxima of
the fluorophores employed in the membrane. The membrane is brought
into contact with an aqueous KCl solution or CaCl.sub.2 solution of
defined concentration by pumping the solution through the cell at a
rate of 1 ml/min and determining the change in fluorescence
intensity. Before the measurement and after each measurement, the
cell is rinsed with potassium ion-free buffer solutions and the
fluorescence intensity is determined in order to define the base
line. The fluorescence intensity (measured as the change in voltage
in the photodiode) in percent at the respective potassium
concentration for the fluorophore of Example A5 (membrane B1) and
various compositions as in Examples C1-C7 is shown in the tables
below.
Example D1 (membrane C2):
______________________________________ Potassium concentration (mM)
Fluorescence (volts) ______________________________________ 0.00
4.32 0.1 3.96 0.5 3.67 1.0 3.52 3.0 3.46 5.0 3.33 7.0 3.23 10.0
3.15 ______________________________________
Example D2 (membrane C6):
______________________________________ Potassium concentration (mM)
Fluorescence (volts) ______________________________________ 0.00
5.30 0.1 3.20 0.5 1.70 1.0 1.20 3.0 0.50 5.0 0.40 7.0 0.30 10.0
0.20 ______________________________________
Example D3 (membrane C1):
______________________________________ Potassium concentration (mM)
Fluorescence (volts) ______________________________________ 0.00
6.89 0.5 4.00 4.0 2.89 10.0 1.89
______________________________________
Example D4 (membrane C4):
______________________________________ Potassium concentration (mM)
Fluorescence (volts) ______________________________________ 0.00
6.80 0.5 4.50 4.0 3.20 10.0 2.50
______________________________________
Example D5 (membrane C5):
______________________________________ Potassium Concentration (mM)
Fluorescence (volts) ______________________________________ 0.00
3.00 0.5 2.40 4.0 2.25 10.0 2.15
______________________________________
Example D6 (membrane C7):
______________________________________ Potassium concentration (mM)
Fluorescence (volts) ______________________________________ 0.00
1.55 0.1 0.96 0.5 0.75 1.0 0.65 3.0 0.50 5.0 0.45 7.0 0.40 10.0
0.38 ______________________________________
* * * * *